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Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

ABSTRACT: How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

SUBMITTER: Travis BA

PROVIDER: S-EPMC9249791 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

Travis Brady A BA Peck Jared V JV Salinas Raul R Dopkins Brandon B Lent Nicholas N Nguyen Viet D VD Borgnia Mario J MJ Brennan Richard G RG Schumacher Maria A MA

Nature communications 20220701 1

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitr ...[more]

PMID: 35778410

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Project description:Paenibacillus polymyxa WLY78, a N2-fixing bacterium, has great potential use as a biofertilizer in agriculture. Recently, we have revealed that GlnR positively and negatively regulates the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) in P. polymyxa WLY78 by binding to two loci of the nif promoter according to nitrogen availability. However, the regulatory mechanisms of nitrogen metabolism mediated by GlnR in the Paenibacillus genus remain unclear. In this study, we have revealed that glutamine synthetase (GS) and GlnR in P. polymyxa WLY78 play a key role in the regulation of nitrogen metabolism. P. polymyxa GS (encoded by glnA within glnRA) and GS1 (encoded by glnA1) belong to distinct groups: GSI-α and GSI-β. Both GS and GS1 have the enzyme activity to convert NH4+ and glutamate into glutamine, but only GS is involved in the repression by GlnR. GlnR represses transcription of glnRA under excess nitrogen, while it activates the expression of glnA1 under nitrogen limitation. GlnR simultaneously activates and represses the expression of amtBglnK and gcvH in response to nitrogen availability. Also, GlnR regulates the expression of nasA, nasD1D2, nasT, glnQHMP, and glnS. IMPORTANCE In this study, we have revealed that Paenibacillus polymyxa GlnR uses multiple mechanisms to regulate nitrogen metabolism. GlnR activates or represses or simultaneously activates and inhibits the transcription of nitrogen metabolism genes in response to nitrogen availability. The multiple regulation mechanisms employed by P. polymyxa GlnR are very different from Bacillus subtilis GlnR which represses nitrogen metabolism under excess nitrogen. Both GS encoded by glnA within the glnRA operon and GS1 encoded by glnA1 in P. polymyxa WLY78 are involved in ammonium assimilation, but only GS is required for regulating GlnR activity. The work not only provides significant insight into understanding the interplay of GlnR and GS in nitrogen metabolism but also provides guidance for improving nitrogen fixation efficiency by modulating nitrogen metabolism.

Project description:Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.

Project description:Bacteria have developed several molecular conflict systems to facilitate kin recognition and non-kin competition to gain advantages in the acquisition of growth niches and of limited resources. One such example is a large class of so-called polymorphic toxin systems (PTSs), which comprise a variety of the toxin proteins secreted via T2SS, T5SS, T6SS, T7SS and many others. These systems are highly divergent in terms of sequence/structure, domain architecture, toxin-immunity association, and organization of the toxin loci, which makes it difficult to identify and characterize novel systems using traditional experimental and bioinformatic strategies. In recent years, we have been developing and utilizing unique genome-mining strategies and pipelines, based on the organizational principles of both domain architectures and genomic loci of PTSs, for an effective and comprehensive discovery of novel PTSs, dissection of their components, and prediction of their structures and functions. In this study, we present our systematic discovery of a new type of PTS (S8-PTS) in several gram-positive bacteria. We show that the S8-PTS contains three components: a peptidase of the S8 family (subtilases), a polymorphic toxin, and an immunity protein. We delineated the typical organization of these polymorphic toxins, in which a N-terminal signal peptide is followed by a potential receptor binding domain, BetaH, and one of 16 toxin domains. We classified each toxin domain by the distinct superfamily to which it belongs, identifying nine BECR ribonucleases, one Restriction Endonuclease, one HNH nuclease, two novel toxin domains homologous to the VOC enzymes, one toxin domain with the Frataxin-like fold, and several other unique toxin families such as Ntox33 and HicA. Accordingly, we identified 20 immunity families and classified them into different classes of folds. Further, we show that the S8-PTS-associated peptidases are analogous to many other processing peptidases found in T5SS, T7SS, T9SS, and many proprotein-processing peptidases, indicating that they function to release the toxin domains during secretion. The S8-PTSs are mostly found in animal and plant-associated bacteria, including many pathogens. We propose S8-PTSs will facilitate the competition of these bacteria with other microbes or contribute to the pathogen-host interactions.

Dataset Information

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

Publications

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

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